| After peripheral nerve injury, axonal degeneration and inflammatory reactions gradually lead to neuronal degeneration and necrosis; Schwann cells and fibroblasts act with each other and form connective tissue scar, obstructing axonal regeneration and arrival at remote target organs. To promote peripheral nerve regeneration effectively and help it arrive at remote target organs before adverse factors appear is actually one of new ideas on the research and treatment of peripheral nerve injury.A large number of experiments in vivo showed that electrical stimulation can effectively promote peripheral nerve regeneration. However, a number of molecular pathways involved in the repair process of damaged nerve induced by electrical stimulation, and the mechanism is very complicated. We have already studied on promotion of brain derived neurotrophic factor (BDNF) secretion in Schwann cells in vitro by using electrical stimulation. But there are lacks of systematic studies in depth on interfering neurons with electrical stimulation cultured in vitro and on the complex mechanisms. For this reason, primary dorsal root ganglion neurons were cultured in vitro, and the electrical stimulation parameters which could effectively promote neurite outgrowth were fully screened. Using this parameter of electrical stimulation to act on neurons, signaling pathways which could involve in the process were verified. This study helps us fully understand the molecular mechanism of peripheral nerve regeneration by providing new experimental evidence and theoretical basis.Objective To screen the electrical stimulation parameters which could effectively promote neurite outgrowth, and to investigate the signaling pathway involved in the promotion of neurite outgrowth by electrical signal.Methods (1) By using serum free Neurobasal/B27 medium to culture dorsal root ganglion neurons in vitro at low density. (2) To stimulate neurons from four aspects, i.e. biphasic/monophasic, duration, voltage, and frequency. After immunocytochemical stain, neurite lengths were measured by using Image-Pro Plus 6.0 software, and screen the parameters which could effectively promote neurite outgrowth. (3) To observe changes of calcium level in neurons by using laser scanning confocal microscope/calcium imaging technologies and pharmacological experiment, and identify sources of increased intracellular calcium. (4) By blocking sources of intracellular calcium, the relationship between calcium level and neurite outgrowth and expressions of c-fos and BDNF at molecular and mRNA levels were studied under electrical stimulation. (5) After calcium-CaM-dependent protein kinases (CaMKs) were inhibited, expressions of phosphorylated cAMP-responsive element binding protein (pCREB), c-fos, and BDNF, and neurite outgrowth were analyzed under electrical stimulation.Results (1) Neurons growed in good condition, and the purity of neurons was (92±6)%. (2) At 1, 3, 5d after electrical stimulation, the parameters of biphasic, 30min, 5V and 10Hz could effectively promoted neurite outgrowth. (3) Under electrical stimulation, increased intracellular calcium derived from two paths. First, L and N type voltage-dependent calcium channels (VDCC) involved in calcium influx from extracellular. Second, IP3 receptor (IP3R) and ryanodine receptor (RyR) calcium stores involved in the process of intracellular calcium mobilization. (4) The promotion of neurite outgrowth and upregulation of pCREB, c-fos, and BDNF expressions induced by electrical stimulation showed obvious calcium-dependent. (5) CaMKs involved in the enhancement of CREB phosphorylation and c-fos and BDNF expressions in neurons induced by electrical stimulation. (6) CaMKs involved in the promotion of neurite outgrowth induced by electrical stimulation.Conclusion Under electrical stimulation, calcium entried through L-VDCC and N-VDCC, and was released from IP3R and RyR calcium stores into cytosol, leading to intracellular calcium elevation. The increasing calcium enhanced the activity of CaMKs, which could phosphorylate CREB, so that CREB was activated. Phosphorylated CREB could act on and increase c-fos and BDNF transcription and translation, which leaded to the upregulation of c-fos and BDNF expressions in neurons, leading to promotion of neurite outgrowth.
|
PDF Full Text Request